The invention relates to DNA polymerases and antimicrobial compounds.
Gram-positive eubacteria include a number of human pathogens, including Staphylococcus aureus, responsible for many human nosocomial soft-tissue infections. Like other common eubacteria, Gram-positive eubacteria absolutely require DNA polymerase III for their growth and replication.
Discovered in 1972, eubacterial DNA polymerase III (pol III) is the major polymerase enzyme involved in DNA replication and is therefore essential for cell division. Two classes of pol IIIs are known.
The Gram-positive pol IIIs are so-named because they were first discovered in the Gram-positive eubacterium Bacillus subtilis. Later it was recognized that Gram-positive pol IIIs are encoded by the polC gene. The polC gene product is generally a polypeptide which is about 1430-1460 amino acids in length, and which integrates both an 3xe2x80x2-5xe2x80x2 exonuclease site and a polymerase site. The Gram-positive pol IIIs are uniquely sensitive to inhibitory dGTP analogs of the so-called xe2x80x9cHPUraxe2x80x9d type (Brown, Proc. Natl. Acad. Sci. USA, 67:1454, 1970).
Gram-negative pol IIIs are so-named because they were first discovered in the Gram-negative bacterium Escherichia coli. The Gram-negative pol IIIs are encoded by the dnaE gene, are typically 1155-1165 amino acids in length, contain only the polymerase site, and are completely insensitive to HPUra-like compounds.
The genomes of Gram-negative eubacteria apparently contain dnaE but not polC. The genomes of Gram-positive eubacteria and mycoplasmas contain both polC and dnaE. The dnaE gene product is required for replication of the Gram-negative bacterial genome, while the polC gene product is required for replication of the Gram-positive and mycoplasmal bacterial genomes. The function of the dnaE gene product in Gram-positive bacteria and mycoplasma is unclear.
The invention is based on the discovery that the DNA polymerase III of Gram-positive eubacteria and mycoplasmas contain a zinc finger domain adjacent to the polymerase active site, and that the integrity of the zinc finger is required for polymerase activity.
Accordingly, the invention features methods of identifying compounds that inhibit infections by Gram-positive eubacteria and mycoplasmas and the new antimicrobial compounds themselves.
In general, the invention features a compound for inhibiting Gram-positive eubacterial or mycoplasmal infection. The compound includes a zinc finger-reactive moiety, a linker, and a Gram-positive eubacterial or mycoplasmal DNA polymerase III active site-binding moiety connected to the zinc finger-reactive moiety via the linker. The compound can have the formula:
Axe2x80x94(Lxe2x80x94B)m
where B is a zinc finger-reactive moiety, L is a linker, and A is a polymerase III active site-binding moiety. Examples of A include: 
in which each of R1 and R2, independently, is hydrogen, C1-3 alkyl, C1-3 haloalkyl, or xe2x80x94Lxe2x80x94B; each of R3 and R4, independently, is hydrogen, C1-3 alkyl, halo, C1-3 haloalkyl, or xe2x80x94Lxe2x80x94B; m is 1 or 2; and n is 0, 1, or 2; provided that at least one of R1, R2, R3, and R4, is xe2x80x94Lxe2x80x94B. The invention also includes salts of the compounds of the invention. L can be a direct bond or a C1-18 alkylene chain. The alkylene chain optionally containing 1 to 5 ether groups, thioether groups, amine groups, ester groups, thioester groups, or amide groups. B can contain an azodi(bis)urea group, an aromatic or aliphatic disulfide group, an aromatic or aliphatic nitroso group, a thiosulfonate group, or a thiazolidone group.
Examples of B include: 
where each of Ra and Rb, independently, is hydrogen, C1-6 alkyl, phenyl, C1-6 hydroxyalkyl, C1-6 haloalkyl, amine, or xe2x80x94Lxe2x80x94A; and p is 1, 2, 3, or 4; provided that either one of Ra and Rb is xe2x80x94Lxe2x80x94A, and Ra and Rb are not xe2x80x94Lxe2x80x94A simultaneously. The invention also includes a salt of any of the above compounds.
In another aspect, the invention includes a method of inhibiting the polymerase activity of a zinc finger-containing DNA polymerase (e.g., a Gram-positive eubacterial DNA polymerase III or a mycoplasmal DNA polymerase III, such as the Bacillus subtilis DNA polymerase III) by contacting the DNA polymerase with a compound (e.g., a compound of the invention) under conditions sufficient for the compound to remove or interacts with a zinc ion bound to a zinc finger in the DNA polymerase.
The invention also includes a method of decreasing the rate of cell division of a bacterium containing a zinc finger-containing DNA polymerase (e.g., a Gram-positive eubacterial DNA polymerase III or a mycoplasmal DNA polymerase III, such as the Bacillus subtilis DNA polymerase III) by exposing the bacterium to a compound (e.g., a compound of the invention) under conditions sufficient for the compound to enter the bacterium and interact with a zinc ion bound to a zinc finger in the DNA polymerase.
In yet another aspect, the invention includes a method for testing whether a compound decreases the rate of cell division of a bacterium (e.g., a Gram-positive eubacterium or a mycoplasma) containing a zinc finger-containing DNA polymerase by exposing a bacterium containing a zinc finger-containing DNA polymerase to the compound under conditions sufficient for the compound to enter the bacterium; and determining whether a zinc ion (e.g., a 65Zn ion) is bound to a zinc finger of the DNA polymerase, where binding of a zinc ion to the zinc finger in the absence of the compound but not in the presence of the compound indicates that the compound decreases the rate of cell division of the bacterium. The zinc finger-containing DNA polymerase can be at least 70% homologous or identical to SEQ ID NO:1 and comprises the sequence:
Zxe2x80x94X2xe2x80x94Cysxe2x80x94X15-27xe2x80x94Cysxe2x80x94X2xe2x80x94Cysxe2x80x83xe2x80x83(SEQ ID NO:5)
where Z is His or, Cys, X2 is any two consecutive amino acids, and X15-27 is any 15 to 27 consecutive amino acids. For example, the zinc finger-containing DNA polymerase can include SEQ ID NO:2. In other embodiments, the zinc finger-containing DNA polymerase comprises the sequence:
CySxe2x80x94X2xe2x80x94Cysxe2x80x94X19-21xe2x80x94Cysxe2x80x94X2xe2x80x94Cysxe2x80x83xe2x80x83(SEQ ID NO:6)
where X2 is any two consecutive amino acids, and X19-21 is any 19 to 21 consecutive amino acids.
The invention also includes a method for testing whether a compound inhibits a zinc finger-containing DNA polymerase by providing a mixture that includes a polypeptide including a zinc finger of a zinc finger-containing DNA polymerase; mixing the compound with the mixture under conditions sufficient to allow the compound to contact the zinc finger; and determining whether a zinc ion is bound to the zinc finger, where binding of the zinc ion to the zinc finger in the absence of the compound but not in the presence of the compound indicates that the compound inhibits the DNA polymerase. In some embodiments, the mixture includes a cell containing the polypeptide.
In a different aspect, the invention includes a method of determining whether a compound inhibits a zinc finger-containing DNA polymerase by providing a mixture that includes a bacterium containing a zinc finger-containing DNA polymerase; mixing the compound with the mixture under conditions sufficient to allow the compound to contact the DNA polymerase within the bacterium, the compound including a group that interacts with zinc in a zinc finger; and measuring polymerase activity of the DNA polymerase in the presence of the compound, where a polymerase activity in the presence of the compound less than the polymerase activity in the absence of the compound indicates that the compound inhibits the DNA polymerase.
In still another aspect, the invention includes a method of treating a mammal susceptible to or having an undesirable Gram-positive eubacterial or mycoplasmal infection by administering to the mammal an amount of a compound (e.g., a compound of the invention) sufficient to interact with zinc in a zinc finger-containing DNA polymerase within a bacterium such that the polymerase activity of the DNA polymerase is inhibited. This method of the invention is especially useful in treating a mammal susceptible to or having an undesirable Gram-positive eubacterial infection.
The invention also includes polypeptides useful in the methods of the invention that include a zinc finger of the sequence CX2CX19-21CX2C (SEQ ID NO:6) or HX2CX21-24CX2C (SEQ ID NO:7), where C is cysteine, H is histidine, X2 is any two consecutive amino acids, X19-21 is any 19 to 21 consecutive amino acids, and X21-24 is any 21 to 24 consecutive amino acids, and can optionally include a polymerase domain. The polypeptides of the invention are shorter than any naturally occurring Gram-positive eubacterial or mycoplasmal pol III.
The new antimicrobial compounds or agents can exist as neutral compounds or salts. For example, the amine groups can be positively charged and form a salt with anions, e.g., bromide. Likewise, any anionic groups of the antimicrobial agent can form a salt with an cation, e.g., a sodium ion, a potassium ion, or an ammonium ion.
Typical alkyl groups are, e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, hexyl, heptyl, octyl, and dodecyl. An alkylene chain is a bivalent alkyl radical.
Halo groups are halogen radicals, e.g., chloro, bromo, or iodo. The halo group in a haloalkyl can attach to any carbon atom of the alkyl group. Likewise, the hydroxy group in a hydroxyalkyl can also attach to any carbon atom of that alkyl group.
When an ester group, a thioester group, or an amide group is present in a new antimicrobial compound, these groups can be connected in either orientation. For example, an ester group can be present as xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94 or xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94.
The nitrogen atom of an amine or an amide can be bonded to a hydrogen or a C1-3 alkyl group.
A xe2x80x9czinc fingerxe2x80x9d is a polypeptide sequence that specifically binds zinc by coordination with (1) four Cys residues, (2) three Cys residues and one His residue, or (3) two Cys residues and two His residues within the polypeptide sequence.
A xe2x80x9czinc finger-reactive moietyxe2x80x9d is a compound or a portion of a compound which, upon contacting a zinc finger, removes the zinc ion from the zinc finger or otherwise interacts with the zinc ion to change the three-dimensional structure of the zinc finger so that an enzymatic activity of a polypeptide containing the zinc finger, e.g., the polymerase activity of pol III, is inhibited.
A xe2x80x9cDNA polymerasexe2x80x9d is a protein or polypeptide that catalyses the polymerization of 2xe2x80x2-deoxyribonucleoside-5xe2x80x2-triphosphates.,
By xe2x80x9cinhibitingxe2x80x9d or xe2x80x9cinhibitedxe2x80x9d is meant partial or complete inhibition.
A xe2x80x9cbacteriumxe2x80x9d is a eubacterium or a member of the order Mycoplasmatales, e.g., a species of the genus Mycoplasma, Spiroplasma, Ureaplasma, or Acholeplasma.
To determine the xe2x80x9cpercent identityxe2x80x9d of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positionsxc3x97100).
The xe2x80x9cpercent homologyxe2x80x9d between two sequences can be determined using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin et al., Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990), modified as in Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol., 215:403-410 (1990). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to T139 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to T139 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers et al., CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment,software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although suitable methods and materials for the practice or testing of the present invention are described below, other methods and materials similar or equivalent to those described herein, which are well known in the art, can also be used. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Among other advantages, the methods of the invention provide a mode of intervention for antibacterial agents which was not previously recognized, namely, antibacterial agents based on the ability to remove or otherwise interact with a zinc ion from a zinc finger within a polypeptide. In addition, the compounds of the invention provide tight specificity for Gram-positive eubacteria and mycoplasmas by combining DNA polymerase III active site-specific chemical groups and zinc finger-reactive chemical groups. Moreover, the present invention provides antimicrobial agents that should be effective against multiple drug resistant (MDR) bacteria, because of their unique and irreversible inhibition of pol III.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
The invention relates to the finding that a unique zinc finger in Gram-positive eubacterial and mycoplasmal DNA polymerase III can be used as a drug target for new antimicrobial compounds. Since disruption of the zinc finger irreversibly inhibits polymerase activity, and such activity is essential for bacterial growth, compounds that specifically disrupt the zinc finger form a new and distinct class of antimicrobial agents. These antimicrobial agents can be used to formulate pharmaceutical compositions suitable for treating mammals (e.g., humans, dogs, cats, horses, cows, and pigs) at risk for or already infected with a Gram-positive eubacterium or mycoplasma. The recognition of the importance of the zinc finger for polymerase activity, and therefore for Gram-positive eubacterial or mycoplasmal growth, also leads to new methods of screening for potential antimicrobial compounds.
I. Discovery of a Zinc Finger in Eubacterial DNA Polymerase III and Mycoplasmal DNA Polymerase
A) Production and Isolation of DNA Polymerase
The polC-specific DNA polymerases useful in the methods of the invention include any naturally occurring Gram-positive eubacterial or mycoplasmal DNA polymerase III. In addition, the invention includes the use of polypeptides having additions or substitutions of amino acid residues within a naturally occurring Gram-positive eubacterial or mycoplasmal pol III. To facilitate production of Gram-positive eubacterial pol III polypeptides, nucleic acids containing the whole or a part of a polC sequence encoding such a pol III can be used for expression. For example, a nucleic acid sequence encoding the Bacillus subtilis pol III is available as GenBank Accession No. X52116. The sequence encodes the following pol III amino acid sequence:
MEQLSVNRRQFQILLQQINMTDDTFMTYFEHGEIKKLTIHKASKSWHFHFQFKSLLPFQIYDTLTTRLTQSFAHIAKVTSSIEVQDAEVSESIVQDYWSRCIEELQGISPPIISLLNQQKPKLKGNKLIVKTKTDTEAAALKNKYSSMIQAEYRQFGFPDLQLDAEIFVSEQEVQKFREQKLAEDQERAMQALIEMEKKDKESDEDQAPSGPLVIGYQIKDNEEIRTLDSIMDEERRITVQGYVFDVETRELKSGRTLCIFKITDYTNSILIKMFAREKEDAALMKSLKKGMWVKARGSIQNDTFVRDLVMIANDVNEIKAKTREDSAPEGEKRVELHLHSPMSQMDAVTGIGKLVEQAKKWGHEAIALTDHAVVQSFPDAYSAAKKHGIKMIYGMEANLVDDGVPIAYNAAHRLLEEETYVVFDVETTGLSAVYDTIIELAAVKVKGGEIIDKFEAFANPHRPLSATIIELTGITDDMLQDAPDVVDVIRDFREWIGDDILVAHNASFDMGFLNVAYKKLLEVEKAKNPVIDTLELGRFLYPEFKNHRLNTLCKKFDIELTQHHRAIYDTEATAYLLLKMLKDAAEKGIQYHDELNENMGQSNAYQRSRPYHATLLAVNSTGLKNLFKLVSLSHIHYFYRVPRIPRSQLEKYREGLLIGSACDRGEVFEGMMQKSPEEVEDIASFYDYLEVQPPEVYRHLLELELVRDEKALKEIIANITKLGEKLNKPVVATGNVHYLNDEDKIYRKILISSQGGANPLNRHELPKVHFRTTDEMLEAFSFLGEEKAKEIVVTNTQKVASLVDDIKPIKDDLYTPKIEGADEEIREMSYQRARSIYGEELPEIVEARIEKELKSIIGHGFAVIYLISHKLVKRSLDDGYLVGSRGSVGSSLVATLTEITEVNPLPPHYVCPECOHSEFFNDGSVGSGFDLPDKTCPHCGTPLKKDGHDIPFETFLGFKGDKVPDIDLNFSGEYQPQAHNYTKVLFGEDNVYRAGTIGTVAEKTAYGYVKGYAGDNNLHMRGAEIDRLVQGCTGVKRTTGQHPGGIIVVPDYMDIYDFSPIQFPADATGSEWKTTHFDFHSIHDNLLKLDILGHDDPTVIRMLQDLSGIDPKTIPTDDPEVMKIFQGTESLGVTEEQIGCKTGTLGIPEFGTRFVRQMLEDTKPTTFSELVQISGLSHGTDVWLGNAQELIHNNICELSEVIGCRDDIMVYLIYQGLEPSLAFKIMEFVRKGKGLTPEWEEEM NNVPDWYIDSCKKIKYMFPKAHAAAYVLMAVRIAYFKVHHALLYYAAYFTVRADDFDIDTMIKGSTAIRAVMEDINAKGLDASPKEKNLLTVLELALEMCERGYSFQKVDLYRSSATEFIIDGNSLIPPFNSIPGLGTNAALNIVKAREEGEFLSKEDLQKRGKVSKTILEYLDRHGCLESLPDQNQLSLF (SEQ ID NO:1)
In the above sequence, the proposed zinc finger amino acid sequence (underlined) is HYVCPECQHSEFFNDGSVGSGFDLPDKTCPHC (SEQ ID NO:2). Twenty four amino acids C-terminal to this sequence is the conserved amino acid sequence PDID (bold) (SEQ ID NO:3). Thus, it appears that the zinc finger is part of the polymerase active site.
Nucleic acid sequences encoding mycoplasmal pol IIIs are also available. For example, the M. pulmonis DNA polymerase sequence is described in the GenBank profile of Accession No. U06833.
In general, the DNA polymerases can be isolated from their natural bacterial sources using standard techniques. Alternatively, the DNA polymerases can be produced by transformation (transfection, transduction, or infection) of a host cell with a DNA polymerase encoding DNA fragment in a suitable expression vehicle. Suitable expression vehicles include plasmids, viral particles, and phage. For insect cells, baculovirus expression vectors are suitable. The entire expression vehicle, or a part thereof, can be integrated into the host cell genome. In some circumstances, it is desirable to employ an inducible expression vector, e.g., the LACSWITCH(trademark) Inducible Expression System (Stratagene; LaJolla, Calif.).
Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems can be used to provide the recombinant protein. The precise host cell used is not critical to the invention.
Preferred DNA polymerases are those which are soluble under normal physiological conditions. Also within the invention are fusion proteins in which a portion (e.g., the zinc finger) of a DNA polymerase is fused to an unrelated protein or polypeptide (i.e., a fusion partner) to create a fusion protein. The fusion partner can be a moiety selected to facilitate purification, detection, or solubilization, or to provide some other function. Fusion proteins are generally produced by expressing a hybrid gene in which a nucleotide sequence encoding all or a portion of DNA polymerase is joined in-frame to a nucleotide sequence encoding the fusion partner. For example, the expression vector pUR278 (Ruther et al., EMBO J., 2:1791, 1983), can be used to create lacZ fusion proteins. The pGEX vectors can be used to express foreign polypeptides as fusion proteins containing glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
A fusion protein can be readily purified by an antibody specific for the fusion protein being expressed. For example, a system described in Janknecht et al., Proc. Natl. Acad. Sci. USA, 88:8972 (1981), allows for the ready purification of non-denatured fusion proteins expressed in human cell lines. In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene""s open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose columns, and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. The same procedure can be used for a bacterial culture.
Alternatively, a DNA polymerase or a portion thereof, can be fused to an immunoglobulin Fc domain. Such a fusion protein can be readily purified using an affinity column.
Both naturally occurring and recombinant forms of DNA polymerases can be isolated to be used in the methods of the invention. Secreted forms can be isolated from culture media, while non-secreted forms can be isolated from the host cells. Further purification can be accomplished by affinity chromatography. In one example, a hexahistidine-tagged derivative of B. subtilis pol III (produced as described herein) is expressed in E. coli. The bacteria is lysed, and the lysate is passed through a Ni-charged IMAC-agarose column (Sigma), which is prepared according to manufacturer""s instructions. The recombinant polymerase is then eluted using an imidazole gradient. Fractions are collected and assayed for polymerase activity. Active fractions are pooled to obtain a mixture containing the polymerase.
Once isolated, the DNA polymerase can, if desired, be further purified and/or concentrated, as long as further processing does not impair the polymerase activity, which can be measured using the procedures described herein. A variety of methods for purification and concentration are well known in the art (see, e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, eds., Work and Burdon, Elsevier 1980), including ultracentrifugation and/or precipitation (e.g., with ammonium sulfate), microfiltration (e.g., via 0.45 xcexcm cellulose acetate filters), ultrafiltration (e.g., with the use of a sizing membrane and recirculation filtration), gel filtration (e.g., columns filled with Sepharose CL-6B, CL-4B, CL-2B, 6B, 4B or 2B, Sephacryl S-400 or S-300, Superose 6 or Ultrogel A2, A4, or A6; all available from Pharmacia), fast protein liquid chromatography (FPLC), and high performance liquid chromatography (HPLC).
B) Determining the Zinc Content of a DNA Polymerase
The zinc content of a DNA polymerase can be determined by methods well known to those skilled in the art of biochemistry. For example, a DNA polymerase produced and isolated by any of the methods described above can be dialyzed in an aqueous solution containing EDTA for a length of time sufficient to scavenge non-specifically bound zinc. It is known that a zinc ion bound to a zinc finger cannot be easily removed with EDTA (Klug et al., FASEB J., 9:597-604, 1995). Standard procedures for preparing a metalloprotein for analysis, including one that contains a zinc finger, are described in Vallee et al., Physiol. Rev., 73:79-118 (1993). Such procedures includes segregation of glassware for use in metalloprotein analysis from other laboratory glassware. Such glassware should be washed only with distilled, deionized water. An isolated DNA polymerase is then subjected to atomic absorption spectroscopy to determine the zinc content.
C. Finding the Zinc Finger
A DNA polymerase that is found to contain a zinc ion may contain a zinc finger. Zinc fingers are extremely diverse in sequence, requiring the presence of only four amino acid residues for coordination of the zinc ion in a stretch of at least 10 consecutive amino acids. The four amino acids are four cysteines, three cysteines and one histidine, or two cysteines and two histidines. Zinc fingers are further described in Klug et al., supra. Although a wide variety of proteins contain a zinc finger, no DNA polymerase, other than the Gram-positive eubacterial DNA pol III, has been definitively found to contain this structure.
It has been found that Gram-positive eubacterial and mycoplasmal pol III contain a zinc finger. The zinc finger sequences among these bacteria are highly homologous and correspond to either a CX2CX19-21CX2C (SEQ ID NO:6) or a HX2CX21-24CX2C (SEQ ID NO:7) zinc finger structure, where the X represents any amino acid and the subscript represents the number of consecutive amino acids. A DNA polymerase useful in the methods of the invention includes the general sequence formula: ZX2CX15-27CX2C (SEQ ID NO:5) or ZX2CX18-24CX2C (SEQ ID NO:8) where Z is Cys or His. Examples within this general formula are CX2CX19-21CX2C (SEQ ID NO:6) and HX2CX21-24CX2C (SEQ ID NO:7).
As an initial evaluation, the amino acid sequence of the DNA polymerase can be aligned with previously described zinc finger sequences (see, e.g., Braithwaite et al., supra) or with the zinc finger sequences described herein. If significant homology between the polymerase sequence and a known zinc finger sequence is found and the critical four amino acids are identified within the context of the generic sequences described herein, then site-directed mutagenesis can be used to mutate any of the critical amino acids to, for example, alanine. If the polymerase zinc finger is authentic and critical to the function of the enzyme, then replacement of any of the critical amino acids should affect the polymerase activity of the enzyme.
Validating that a functional (i.e., required for polymerase activity) zinc finger exists in an essential DNA polymerase of a pathogenic organism provides a new drug target for inhibiting the growth of that organism.
II. General Methodology for Disrupting a Zinc Finger in a DNA Polymerase
A) Zinc Finger-Reactive Moieties
By identifying a functional zinc finger in a DNA polymerase, one skilled in the art can inhibit the polymerase by changing the structure of the zinc finger (e.g., by ejecting zinc), thereby inhibiting the growth of an organism that relies on the DNA polymerase for replication. Changes in zinc finger structure can be induced by contacting the zinc finger with a compound that is known to react or interact with a zinc finger. Alternatively, the compound is not initially known to alter zinc finger structure but rather is selected from a library of compounds screened against a zinc finger of the present invention for this;activity. Such compounds are well known in the art, including those described in Rice et al., J. Med. Chem., 39:3606-3616 (1996); Otsuka et al., J. Med. Chem., 37:4267-4269 (1994); Otsuka et al., J. Med. Chem., 38:3264-3270 (1995); Fujita et al., J. Med. Chem., 39:503-507 (1996); Loo et al., J. Med. Chem., 39:4313-4320 (1996); Jaffe et al., J. Biol. Chem., 259:5032-5036 (1984); and Louie et al., Proc. Natl. Acad. Sci. USA, 95:6663-6668 (1998). A more detailed discussion of zinc ejectors appears below.
B) Targeting a Zinc Finger-Reactive Moiety to the DNA Polymerase Active Site
To increase the specificity of zinc finger-reactive moieties for Gram-positive eubacterial or mycoplasmal pol III, any of the zinc finger-reactive moieties described herein can be linked, e.g., covalently linked, to a compound known to bind to the polymerase active site of those DNA polymerases (e.g., the HPUra-like compounds disclosed in U.S. Pat. No. 5,516,905). A zinc finger-reactive moiety brought to such close proximity to the zinc finger via an active site-binding component is expected to increase specificity or potency of the antimicrobial compounds of the invention.
For example, HPUra-like compounds are a class of uracil-based microbial agents that specifically target Gram-positive eubacterial and mycoplasmal DNA pol III by binding to a portion of the polymerase active site. This portion is less than 24 amino acids away from the C-terminus of the zinc finger domains identified herein, and so is in close spatial proximity to the zinc finger.
Thus, by linking a HPUra-like compound to a compound known to react with a zinc finger., a new class of antimicrobial agents specific for Gram-positive eubacteria and mycoplasma are produced. Further details on the synthesis of these new antimicrobials are given below.
III. Screening for Candidate Antimicrobial Compounds
The recognition that an authentic zinc finger is presented in Gram-positive eubacterial and mycoplasmal pol III forms a basis for a new class of antimicrobials against these microorganisms. Thus, candidate compounds (e.g., compounds from a chemical library) can be initially screened for antimicrobial activity by using relatively inexpensive and microarrayable zinc binding or ejection as a surrogate activity. Several screening procedures are described below.
A) Measuring Zinc Released from a DNA Polymerase
A candidate antimicrobial can be tested for its ability to eject zinc from a DNA polymerase by a variety of methods. For example, a bacterium producing a DNA polymerase useful in the methods of the invention can be grown in media free of environmental zinc and supplemented with radioactive 65Zn.
The zinc-free media can be prepared by first mixing a sufficient amount of Chelex-Na (Bio-Rad) into the media for a time sufficient to remove environmental zinc. The Chelex is removed from the media, and [65Zn]Cl2 is added to the media to about 0.5 to 2 xcexcCi/ml media. The bacteria are then grown in this labeling media, and the radioactive zinc-labeled DNA polymerase is isolated and purified using the methods described above. Preferably, zinc which is non-specifically bound to the DNA polymerase is removed by the dialyzation process described above.
The candidate antimicrobial compound can be added to an aqueous mixture or solution of the isolated, zinc-labeled DNA polymerase under conditions that allow contact between the zinc finger of the polymerase and the compound. For ease of measurement, the polymerase is optionally attached to a solid support, e.g., a Sepharose bead. If the compound is effective in ejecting zinc from the zinc finger, radioactivity is released into the solution and lost from the protein. Either the radioactivity level of the protein or the radioactivity of the protein-free solution can be counted by standard methods to determine if the compound is effective. The compound is considered effective in ejecting zinc if at least 25% of the specifically bound zinc is removed by the compound. Preferably at least 50% (e.g., at least 75%, 90%, or 95%) of the zinc is removed.
B) Measuring Polymerase and Exonuclease Activities
After passing the initial screen, a candidate antimicrobial can also be screened for its ability to inhibit a DNA polymerase activity. The effect of the compound on exonuclease activity, as well as polymerase activity, can be measured.
Polymerase activity can be measured by any number of methods well known in the art, e.g., the method described in Barnes et al., Meth. Enzymol., 262:35-42 (1995). Briefly, five microliters of an appropriate dilution of enzyme is rapidly mixed with 20 xcexcl of polymerase assay mix (18.75 mM Tris [pH 7.5], 12.5 mM magnesium acetate, 31.25 xcexcm DATP, 31.25 xcexcm dCTP, 31.25 xcexcm dGTP, 12.5 xcexcm [methyl-3H]dTTP [1.5 xcexcCi/xcexcmol], 1.25 mM DTT, 20% glycerol, and 0.5 mg/ml activated DNA), and incubated at 30xc2x0 C. for 10 minutes. Reactions are stopped by addition of 0.5 ml cold 10% trichloroacetic acid (TCA) in 10 mM sodium pyrophosphate. After approximately 10 minutes at 0xc2x0 C., samples are filtered on Whatman GF/A filters and washed, first with cold 1 M HCl in 100 mM sodium pyrophosphate, then with cold ethanol. Filters are dried and their radioactivity quantitated by liquid scintillation counting.
For determination of the Michaelis constant (KM) of the polymerase for DNA, the concentration of activated calf thymus DNA is varied during the assay from 0.0 to 0.8 mg/ml. For determination of the KM for dGTP, incorporation of [3H]dTMP can be followed as a function of dGTP concentration (e.g., from 0.0 to 0.5 mM), and the values for incorporation are corrected for dGTP-independent background incorporation.
Exonuclease activity also can be measured by methods well known in the art, including those described in Barnes et al., supra. For example, five microliters of an appropriate dilution of enzyme is quickly mixed with 45 xcexcl of exonuclease assay mix (33.3 mM Tris [pH 7.5], 7.4 mM magnesium chloride, 3.3 mM DTT, 11.1% glycerol, and 3H-labeled denatured DNA [0.05-0.2 xcexcg/xcexcl; about 70,000 cpm/assay]), and incubated at 30xc2x0 C. for 10 minutes. Reactions are stopped by addition of 0.5 ml 10% TCA in 10 mM sodium pyrophosphate. Fifty microliters of a 10 mg/ml solution of bovine serum albumin is added as a coprecipitant. After about 10 minutes at 0xc2x0 C., samples are centrifuged at 15,000 g for 20 minutes. Then 400 xcexcl of the supernatant is removed and assayed for radioactivity in 2 ml of an aqueous scintillant. If the presence of the compound in the reaction leads to a substantial decrease in the exonuclease activity, the candidate compound is effective in inhibiting exonuclease activity.
For determination of the KM for the exonuclease substrate, the concentration of single-stranded DNA is varied from 0.0 to 0.2 mg/ml.
C) Measuring Bacteriocidal Activity
A candidate antimicrobial compound can be screened for its ability to decrease the rate of cell division of a bacterium (bacteriostatic and/or bacteriocidal activity). Methods of measuring the rate of cell division are well known in the art. For example, the rate of cell division can be measured by counting the difference in cell number at two time points, taking the log2 of that difference, and dividing that value by the time elapsed between the two time points. If the measured rate of cell division of a bacterium grown in the presence of the compound is substantially less than in the absence of the compound, the candidate compound is effective in decreasing the rate of cell division.
As an example of a primary screen, the candidate antimicrobial compound is dissolved in sterile DMSO and diluted 100-fold (final DMSO concentration of 1%) into Mueller-Hinton broth (MHB; Difco) containing log-phase methicillin-sensitive S. aureus (ATCC No. 29213) at about 106 colony forming units (cfu) per milliliter. The control culture contains only 1% DMSO. Compound and control cultures are incubated at 37xc2x0 C., and samples from the cultures are removed at specific times during the next 24 hours. Each sample is assayed for bacteria in cfu/ml by diluting in MHB and plating on LB agar plates. The candidate compound is said to have bacteriocidal activity if the cfu/ml of the relevant sample is reduced by at least 50% in the presence of:the compound as compared to in the absence of the compound.
To determine if any of the compound-exposed bacteria has developed resistance to the antimicrobial compound, bacteria are grown for three days in medium containing a concentration of the compound which still allows at least some growth. This;bacteria is used in a secondary bacteriocidal activity assay (same procedure as above). Resistance is indicated if the decrease in cfu/ml seen in the secondary assay is substantially less than the decrease in cfu/ml seen in the primary assay. Alternatively, 108 cfu of bacteria is plated on 150 mm petri plates containing 3xc3x97, 10xc3x97, or 30xc3x97 MIC of the antimicrobial compound. After incubation at 37xc2x0 C. for three days, colonies are counted and related to the number of cells plated to give an estimate of the mutation frequency.
D) Minimal Inhibitory Concentration (MIC)
To determine minimal inhibitory concentration of a candidate antimicrobial compound, log-phase bacterial cultures are diluted to about 104/ml in LB medium containing 1% DMSO. 0.5 ml of the suspension is distributed to each well of a 48-well microtiter plate. The compound is added to the wells to achieve 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.575, or 0 micromolar concentrations of the compound. The plate is incubated for 24 hours at 37xc2x0 C. and read by visual inspection of the wells. The minimal inhibitory concentration (MIC) is defined as the lowest concentration of inhibitor at which bacterial growth was not visually apparent.
Alternatively, MIC can be determined as follows. Stock solutions of the compound is added to individual containers of liquid LB media containing 1.4% agar at 60xc2x0 C. to achieve compound concentrations of 48, 24, 12, 6, 3, 1.5, 0.75, 0.375, and 0.19 xcexcg/ml. The LB agar is poured onto petri plates and solidified. 100 xcexcl of about 500 to 1000 cfu is plated onto each petri plate, including a control plate without compound. The plates are incubated at 37xc2x0 C. for 24 hours. MIC is determined as the lowest concentration at which no colony formation is observed.
E) In Vitro Cytotoxicity Screening
A candidate antimicrobial compound also can be screened for in vitro cytotoxicity. At various concentrations, the compound is added to small spinner cultures of exponentially growing mammalian cells (e.g., HeLa S3). At 8 hour intervals for the next 48 hours, samples are taken from the cultures and the cell number counted by standard, techniques (e.g., Coulter counting). Preferably, the compound is assessed at 3xc3x97 and 10xc3x97 the MIC concentration (see above).
F) In Vivo Lethal Protection Screening
To determine if a candidate antimicrobial compound can protect an animal from a lethal bacterial challenge, 20-gram female Swiss-Webster mice are infected with a single intraperitoneal (ip.) injection of methicillin-sensitive S. aureus xe2x80x9cSmithxe2x80x9d strain (0.5 ml in physiological saline; 4xc3x97107 cfu/mouse). One hour later, the mice are individually injected with various solutions/suspensions. The negative control mouse receives 0.1 ml of physiological saline. The positive control mouse receives 0.1 ml of a 4 mg/ml solution of vancomycin in saline, which corresponds to a dose of 20 mg/kg body weight. The test mouse receives about 1 to 10 mg/kg of the compound in an appropriate diluent. If the compound diluent is not saline, then another mouse is injected with the compound diluent as a second negative control. Each mouse is monitored for survival over a three day period. The compound is said to protect against this lethal challenge if the mouse injected with the compound lives or vancomycin lives, but the mouse receiving the diluent dies at the end of the observation period.
The protection screening can be performed by a commercial subcontractor, e.g., MDS Panlabs, Inc.
G) In Vivo Acute Toxicity Screening
The in vivo acute toxicity of a candidate antimicrobial compound can be determined. Various concentrations of the compound are administered to the tail vein of mice. Each mice receives 0.05 to 0.2 ml inoculum containing 25, 50, 100, or 150 mg compound/kg body weight. The mice are observed closely for 12 hours for signs of acute toxicity, such as lethargy, shivering, tendency to immobility, or hunchbacking. Doses which cause more than temporary discomfort are noted. All animals used in the study are euthanized by decapitation at the end of the observation period.
H) In Vivo Half Life
The intravenous in vivo half life of a candidate antimicrobial compound can be estimated. Mice are injected, via the tail vein, :with the highest dose that does not cause acute toxicity (see above). At 10, 20, 30, 45, 90, and 150 minutes after injection, two mice are decapitated and their blood collected by exsanguination into a sterile test tube. The blood samples are centrifuged, and the plasma collected. 0.2 ml of the plasma is used for HPLC analysis to determine the amount of compound in the blood at the indicated time after injection.
The half-life is determined by noting the time required for the blood compound level to reach 50% of any previously amount noted for a specific time, with the proviso that time points are taken during the decay phase of the blood compound levels. In other words, the maximum blood compound level is achieved before any timed sample is taken for the purpose of determining the half-life.
Alternatively, other tissues besides blood can be evaluated for compound levels after administration. For example, instead of collecting the blood from sacrificed mice, the liver can be collected, homogenized, cleared, and assayed for compound levels. The compound levels and half-lives in various tissues are useful for determining the tissue distribution of the compound and any variances between the blood compound levels and levels in other tissues under one method of administration.
Such results are also important in determining any pharmacological differences associated with a specific route of administration. For example, the compound could have a dramatically higher bioavailability in the lung when administered by inhalation than when administered subcutaneously.
I) In Vivo Efficacy Screening Using the Thigh Infection Mouse Model
In vivo efficacy screening also can be performed using the thigh infection model described below. This model is rational, flexible, relatively inexpensive, and reproducible. It is also well described in the art (see, e.g., Gudmundsson et al., J. Antimicrob. Chemother., 31:177-191, 1993).
In the thigh infection mouse model, mice are made neutropenic (e.g., by administering cyclophosphamide to the mice) to render them susceptible to infection with a wide variety of bacteria. The mice are then infected by intramuscular (im). injection of test bacteria (one or more species) into the thigh. The infected mice are typically divided into at least three groups., The first group receives treatment with the candidate antimicrobial compound. A second group receives a known efficacious antibiotic (e.g., vancomycin). The third group receives only the diluent used to deliver the compound and antibiotic, if the diluent is the same in both cases. If the diluents used for the compound differs from that used for the vancomycin, another control group may be necessary to test the effect of the second diluent.
Just before the treatment begins, and at predetermined times after infection, animals are sacrificed. The portion of the thigh into which the bacteria had been injected is removed, homogenized in sterile saline, diluted, and plated onto standard bacterial agar plates to determine the bacterial content in cfu/ml.
Typically, the infection is designed to avoid death of untreated animals in the period of experimental observation. Death can be avoided if the inoculum and the period of observation are chosen such that the number of bacteria in the thigh of an untreated, infected animal increases by no more than two to three logs. The efficacy of the compound is typically based on the capacity of a given dose to prevent this increase and to reduce the bacterial load to lower than 50% of the load which is present in the diluent-treated animal. In this assay, 40 mg/kg vancomycin given intravenously every four hours produces a range of two to four log reduction in S. aureus proliferation compared with control thighs.
The choice of diluents and route of administration will be dictated primarily by the physiochemical properties of each candidate compound. For compounds that have significant solubility in water, dissolution and administrating in saline by any route is possible. More hydrophobic compounds may require a diluent of a mixture of DMSO and water (e.g., 80% DMSO [v/v]), or alternatively 90% peanut oil in DMSO for intraperitoneal administration. For subcutaneous administration, poorly soluble compounds can be micronized/solubilized in a mixture of glycerol, propylene glycol, and water.
IV. Pharmaceutical Compositions and Their Administration
The antimicrobial compounds of the invention can be formulated into pharmaceutical compositions suitable for administration into animals, especially humans.
A) New Antimicrobial Compounds
The new antimicrobial compounds typically contain three components: a Gram-positive eubacterial or mycoplasmal DNA polymerase III active site-binding moiety (xe2x80x9cAxe2x80x9d), a zinc finger-reactive moiety (xe2x80x9cBxe2x80x9d), and a linker (xe2x80x9cLxe2x80x9d) which joins the pol III active site-binding moiety and the zinc finger-reactive moiety together. The new antimicrobial compound is represented by a general formula: Axe2x80x94(Lxe2x80x94B)m. As the novel antimicrobial compounds can contain up to 2 zinc finger-reactive moieties, m can be a positive integer 1 or 2.
The pol III active site-binding moiety can be modified from a compound known to bind to such an active site, e.g., one of the HPUra-like compounds having a formula as shown below: 
Each of R1 and R2, independently, is hydrogen, C1-3 alkyl, C1-3 haloalkyl, or xe2x80x94Lxe2x80x94B. Each of R3 and R4, independently, is hydrogen, C1-3 alkyl, halo, C1-3 haloalkyl, or xe2x80x94Lxe2x80x94B; and n is 0, 1, or 2; provided that at least one of R1, R2, R3, and R4, is xe2x80x94Lxe2x80x94B.
The linker can be as short as a direct bond or as long as a C18 alkylene chain. When the linker is an alkylene chain, it can optionally contain ether, thioether, amine, ester, thioester, or amide. For instance, the alkylene chain can contain multiple (e.g., 1-5) amine groups. A suitable example would be a xe2x80x94(CH2)2xe2x80x94NHxe2x80x94(CH2)3xe2x80x94NHxe2x80x94(CH2)2xe2x80x94 group. The linker can also be a branched alkylene chain, e.g., a xe2x80x94CH(xe2x80x94(CH2)3xe2x80x94)xe2x80x94Oxe2x80x94(CH2)3xe2x80x94Oxe2x80x94(CH2)2xe2x80x94 group, which can be attached to more than one zinc finger-reactive moiety. The ether, thioether, amine, ester, thioester, or amide group can also be present at the ends of the linker, thus joining the other two moieties to the linker.
The zinc finger-reactive moiety can be modified from a zinc finger-reactive group well known in the art, e.g., an azodi(bis)urea group, an aromatic or aliphatic disulfide group, an aromatic or aliphatic nitroso group, a thiosulfonate group, or a thiazolidone group. Such moieties eject or otherwise interact with the zinc ion from the zinc finger by either forming bonds with the zinc ion directly or bonding with the amino acid residues, e.g., cysteine or histidine residues, that coordinate with the zinc ion. Note that the word xe2x80x9cbondxe2x80x9d here can be any form of linkage such as a covalent bond, an ionic bond, or a hydrogen bond. See, e.g., Rice et al., J. Med. Chem., 39:3606-3616 (1996); Otsuka et al., J. Med. Chem., 37:4267-4269 (1994); Otsuka et al., J. Med. Chem., 38:3264-3270 (1995); Fujita et al., J. Med. Chem., 39:503-507 (1996); Loo et al., J. Med. Chem., 39:4313-4320 (1996); Jaffe et al., J. Biol. Chem., 259:5032-5036 (1984); and Louie et al., Proc. Natl. Acad. Sci. USA, 95:6663-6668 (1998).
A novel antimicrobial compound can be prepared by following the general procedure as set forth below.
B) Methods of Preparing New Antimicrobial Compounds
There exist many different routes for the preparation of the new antimicrobial compounds. The following general procedure is not limiting.
Preparation of the new antimicrobial compounds can begin with coupling a pol III active site-binding moiety (xe2x80x9cAxe2x80x9d) to a linker (xe2x80x9cLxe2x80x9d). Methods of preparing 3-substituted pyrimidines and 7- and 9-substituted purines are described in detail in U.S. Pat. Nos. 5,516,905 and 5,646,155, respectively. The substituents can be further modified to form a linker moiety containing a functional group at its terminus for coupling to the zinc finger-reactive moiety (xe2x80x9cBxe2x80x9d). Suitable linker terminal functional groups include typical leaving groups for substitution reactions, e.g., halides; amine groups for forming amide linkages with activated carboxylic acid derivatives, e.g., acid halides; or thio groups for forming disulfide linkages with other thio-containing compounds. The following schemes exemplify the preparation of various novel antimicrobial compounds.
Preparation of Lxe2x80x94A with a Suitable Terminal Functional Group for Coupling to B As described above, the moiety L can be modified to form a functional group for coupling to B. Three examples of such a functional group are illustrated below, i.e., xe2x80x94I (compound I), xe2x80x94SH (compound II), and xe2x80x94NH (compound III). 
Coupling of Lxe2x80x94A to B
The functional group on the moiety L can then be coupled e.g., by a coupling reaction such as alkylation, with B to yield the new antimicrobial compound. Exemplary coupling reactions are described below.
Reaction A
In reaction A, compound I undergoes a nucleophilic substitution to effect an amine linkage between A-L and B, and displaces the iodide as a leaving group. 
Reaction B
In reaction B, the thiol group of compound II displaces the methylsulfonyl group to form a disulfide linkage in the final antimicrobial compound. Similarly, a sulfonatethio linkage forms in the product as the chloride is displaced by the thiol group of compound II in reaction C. 
Reactions D, E, and F
In reactions D, E, and F, the amine group of compound III reacts with an acid chloride, thereby forming an amide linkage. The nitrogen atom of the amide in reaction E further attacks the disulfide bond of moiety B and results in a formation of a ring. 
C) Formulation
The compositions can be formulated as a solution, suspension, suppository, tablet, granules, powder, capsules, ointment, or cream. In the preparation of these compositions, at least one pharmaceutical carrier can be included. Examples of pharmaceutical carriers include solvent (e.g., water or physiological saline), solubilizing agent (e.g., ethanol, polysorbates, or Cremophor EL(copyright)), agent for making isotonicity, preservative, antioxidizing agent, excipient (e.g., lactose, starch, crystalline cellulose, mannitol, maltose, calcium hydrogen phosphate, light silicic acid anhydride, or calcium carbonate), binder (e.g., starch, polyvinylpyrrolidone, hydroxypropyl cellulose, ethyl cellulose, carboxy methyl cellulose, or gum arabic), lubricant (e.g., magnesium stearate, talc, or hardened oils), or stabilizer (e.g., lactose, mannitol, maltose, polysorbates, macrogols, or polyoxyethylene hardened castor oils) can be added. If necessary, glycerin, dimethylacetamide, 70% sodium lactate, a surfactant, or a basic substance such as sodium hydroxide, ethylenediamine, ethanolamine, sodium bicarbonate, arginine, meglumine, or trisaminomethane is added. Biodegradable polymers such as poly-D,L-lactide-co-glycolide or polyglycolide can be used as a bulk matrix if slow release of the composition is desired (see e.g., U.S. Pat. Nos. 5,417,986, 4,675,381, and 4,450,150). Pharmaceutical preparations such as solutions, tablets, granules or capsules can be formed with these components. If the composition is administered orally, flavorings and colors can be added.
The concentration of the compound in the compositions of the invention will vary depending upon a number of factors, including the dosage to be administered, and the route of administration.
D) Administration
The compounds and compositions of the invention can be administered by parenteral administration, for example, intravenous, subcutaneous, intramuscular, intraorbital, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, topical, intranasal, aerosol., scarification, and also oral, buccal, rectal, vaginal, or topical administration. The compositions of the invention may also be administered by the use of surgical implants which release the compounds of the invention.
In general terms, the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral administration, typically after determining whether the patient is susceptible to or having a Gram-positive eubacterial or mycoplasmal infection. General dose ranges are from about 0.01 mg/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 5 mg/kg to 100 mg/kg of body weight per day. The preferred dosage to be administered will depend upon the type and extent of progression of the infection being addressed, the overall health of the patient, and the route of administration. For. topical and oral administration, formulations and dosages can be similar to those used for other antibiotic drugs, e.g., erythromycin or vancomycin.